Streptomyces griseus Aminopeptidase (SGAP)

Title : Structure of S. griseus aminopeptidase

Biological function
S. griseus Aminopeptidase (SGAP; E.C. 3.4.11.-) cleaves the N-terminal amino acid from a peptide or protein, and is specific for larger hydrophobic acids, especially leucine. No cleavage occurs if the next residue is proline.



Biological Context
SGAP is one of the many proteinases present in the extracellular fluid of cultures of Streptomyces griseus, and can be isolated from Pronase, the commercial preparation of the extracellular fluid from this organism. SGAP is a monomeric, 30KDa, heat stable enzyme requiring two Zn2+ ions for activity, and is activated by Ca2+.

Structures Available

 * 1xjo - native SGAP to 1.75Å
 * 1cp7 - native SGAP to 1.58Å
 * 1qq9 - SGAP complexed with L-methionine to 1.53Å
 * 1f2o - complex with L-leucine to 1.70Å
 * 1f2p - complex with L-phenylalanine to 1.80Å
 * 1tf8 - complex with L-tryptophane to 1.30Å
 * 1tf9 - complex with p-iodo-L-phenylalanine to 1.30Å
 * 1tkf - with D-tryptophan to 1.20Å
 * 1tkh - with D-phenylalanine to 1.25Å
 * 1tkj - with D-methionine to 1.15Å
 * 1xbu - with p-iodo-D-phenylalanine to 1.20Å

Historical Context
The proteolytic activity contained in the extracellular fluid of cultures of Streptomyces griseus was first identified by Nomoto and Narahashi (1959a), who obtained a highly purified preparation of this activity from the K-1 strain of this bactreria. A large scale version of their procedure was used to prepare commercial quantities of this preparation (Pronase). Various physical criteria showed that Pronase was homogeneous (Nomoto and Narahashi, 1959b), yet displayed both exopeptidase and endopeptidase activity, with a wide range of side chain specificities (Nomoto and Narahashi, 1959b, 1959c; Nomoto et al., 1960a, 1960b, 1960c). The supposed homogeneity of Pronase was controversial, with other investigators using various chromatographic methods to isolate more fractions with proteolytic activity (Hiramatsu and Ouchi, 1963; Nomoto et al., 1964). Subsequently Narahashi and Yanagita (1967) identified several distinct proteolytic activities including one which had aminopeptidase activity, and was activated by Ca2+ and Co2+. In contrast to other proteinase activities in the mixture, this aminopeptidase activity displayed considerable heat stablity (up to 80℃) and was unaffected by 9M urea. The activity, was, however, very sensitive to metal chelating agents.

While attempting to isolate the protein responsible for the trypsin activity in Pronase, Vosbeck et al. (1973) isolated two fractions with aminopeptidase activity. Although the two fractions differed slighty in their molecular weights (23K and 25K), they appeared to have the same enzymatic properties.

Interest in SGAP was renewed when aminopeptidases were recognized as useful tools in assays of metalloendopeptidase activity. The assay was based on a two stage reaction, with the endopeptidase cleaved an N-blocked peptide to release smaller peptide that was rapidly degraded by an aminopeptidase, generating a chromophore (Orlowski and Wilk, 1981; Mumford et al., 1981). SGAP was considered an ideal tool for this purpose, given its stability, small size, and availability (Indig et al., 1990).